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Journal of Applied Biological Sciences 10 (3): 43-46, 2016
ISSN: 1307-1130, E-ISSN: 2146-0108, www.nobel.gen.tr
Isolation of Atopobium vaginae in Vaginal and Urine Samples of Iranian Women, the first
report
Hamideh MAFTOON1 Nour AMIRMOZAFARI*2 Maryam KASHANIAN3
1Microbiology Department, School of Medicine, International campus, Iran University of Medical Sciences, Tehran, Iran
2Microbiology Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
3Professor of Obstetrics and Gynecology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
*Corresponding Author: Received: August 17, 2016
E-mail:amirmozafari@yahoo.com Accepted: December 20, 2016
Abstract
Bacterial vaginosis is not a mono-factorial infection. A synergism of microaerophilic bacteria, Mycoplasma spp., and anaerobic bacteria such as
Atopobium vaginae, Porphyromonas spp., Bacteroides spp., Prevotella spp., and others are involved in these infections. The aim of present study
was to determine the prevalence of Atopobium vaginae in non-pregnant women suffering from bacterial vaginosis.A total of 102 non-pregnant
women who referred to Shahid Akbarabadi hospital in Tehran were tested for bacterial vaginosis. Bacterial culture was performed on Columbia
agar containing 2mg/ml Amphotericin B, 30mg/ml Nalidixic acid and 4mg/ml Colistin. Additionally, they were simultaneously cultured on
blood agar plates containing fresh human blood and Amphotericin B under anaerobic conditions. Finally, PCR using Atopobium vaginae specific
primers were also carried out on extracted DNA from grown colonies as well as from vaginal specimens. From a total of 102 symptomatic
women who referred to the hospital, 37% (38/102) were classified as bacterial vaginosis, 43% (44/102) were classified as intermediate status and
20% (20/102) women were asymptomatic. Sixty urine samples were collected. Atopobium vaginae were detected in 66% (25/38) of bacterial
vaginosis cases and in 10% (4/44) samples with intermediate vaginal flora. It was not detected in asymptomatic women. The bacterium was seen in
10% (3/30) women with urinary tract infection and none in pregnant women. This is the first report of Atopobium vaginae isolation in Iran. The
results of this investigation point to a clear association of Atopobium vaginae with bacterial vaginosis. It should therefore be considered as a
probable etiological agent.
Keywords: Bacterial vaginosis, Atopobium vaginae, Culture, PCR
INTRODUCTION
Bacterial vaginosis (BV) is considered to be the most
common cause of vaginal disorder and imbalance affecting
women during their reproductive age[1,2,3,4]. This dis-
order is strongly associated with alteration in vaginal mi-
croflora when hydrogen peroxide-producing Lactobacilli
are decreased and replaced by bacteria such as Atopobium,
Prevotella spp., Mobillincus spp., Gardnerella vaginalis and
Bacteroides spp.[4,5,6].The most common bacteria isolated
from BV is Gardnerella vaginalis[6]that grows under appro-
priate microaerophilic condition and the anaerobic bacte-
rium Atopobium vaginae. Despite the first work of Leopoldo
in 1953 and Gardner and Dukes in 1955 [7], not a singlei-
nfectious agent has been associated as the sole causative
agent BV. Ethiopathogenesis of BV is still not fully eluci-
dated [4,7,8].
Bacterial vaginosis is associated with the presence of
a dense and lower case for Polymicrobial biofilm, mainly
populated by Gardnerella vaginalis with strong adhesion to
vaginal epithelium. Bacterial vaginosis is characterized by
elevation of vaginal pH and clinical signs such as foamy vag-
inal discharge, presence of clue cells in wet mount, and fishy
odor (positive KOH amine test) [9,10,11]. Based on Amsel
criteria, the presence of three of these signs is considered to
be a strong indication of BV. However, some patients with-
out BV may reveal similar clinical findings, such as those
with Trichomonas vaginalis[10,11,12]. Bacterial vaginosis
has also been associated with a long list of complications;
such as pelvic inflammatory disease, spontaneous abortion,
preterm birth and low-birth-weight infants[1,2,3,4,13]. This
study was undertaken to detect the extent of Atopobium vagi-
nae associated BV in non-pregnant women from different age
groups who referred to a large general hospital in the Iranian
capital, Tehran. The existence of this bacterium in the urine
samples of pregnant and non-pregnant women was also in-
vestigated.
METHODS
Patients and clinical samples
From November 2014 till October 2015, vaginal samples
from 102 non-pregnant women with clear clinical symptoms
women of reproductive age were collected. No women re-
ceived antimicrobial therapy for at least a week before prior
to sample collection. Urine samples were also collected from
30 non-pregnant women with urinary tract infection as well as
from 30 healthy pregnant women.
Gram stain, Whiff test and pH Measurement
Vaginal discharge was collected in the following order by a
gynecologist: vaginal swabs for direct PCR, which were
placed in PBS. Another vaginal swab for Gram stain and ob-
servation of clue cells, and another sample for determination
of vaginal pH and application of 10% KOH for detection of
amine odor (Whiff test).
Diagnosis of BV
The Amsel criteria were used for diagnosis of BV. Sam-
ples were classified as BV if three or four of the Amsel crite-
ria were present. The samples were categorized as healthy if
less than three of these criteria were seen.
Culture
Atopobium vaginae is an anaerobic facultative bacte-
rium, which was grown on Columbia blood agar supple-
mented with 2mg/ml Amphotericin B, 30mg/ml Nalidixic
acid and 4mg/ml Colistin in an anaerobic chamber (Gas
Pack) at 37˚C for 48-72 hours. The grown colonies were
44
N. Amirmozafari et al /JABS, 10 (3): 43-46, 2016
grey-white in color and non-hemolytic. Additionally, they
were simultaneously cultured on blood agar plates containing
fresh human blood and 2mg/ml Amphotericin B under
anaerobic conditions. Upon staining, Atopobium vaginae
appear as Gram positive, small cocci, and arranged in single
cells, in pairs or short chains. Figure 1. Sequence confirm for Atopobium vaginae
DNA isolation
Total DNA from samples, either grown bacterial colo-
nies or vaginal swabs placed in PBS, were isolated using the
YTA Genomic Extraction Mini Kit #YT9040 (Yakta Tajhiz
Azma, Tehran, Iran) according to the manufactures guide-
lines.
Polymerase chain reaction (PCR) assay
A species-specific PCR assay for the detection of Atopo-
bium vaginae targeting the 16S rRNA gene was arranged [8].
Of the clinical criteria for bacterial vaginosis, homog-
enous discharge was present in 31 (82%) while a pHof ≥
4.5 was found in 34 (89%) cases of bacterial vaginosis.
Amine test was positive in all 38 (100%) cases. Clue cells
were present in 27 (71%) cases of bacterial vaginosis on wet
mount (Table 1).
Table 1.
Bacterial vgainosis based on Nugent score and
prevalence of Atopobium vaginae.
The oligonucleotides used as primers for amplification were
AV1-F (5′-TAGGTCAGGAGTTAAATCTG-3′) and AV3-
R (5′-TCATGGCCCAGAAGACCGCC-3′) [8]. They were
verified for specificity using the BLAST program. The PCR
reaction was carried out in a total of volume of 25.0 µl, and
the final concentration of the mix for each sample contained:
10 µl Master Mix (containing Taq DNA polymerase, dNTPs,
and PCR buffer), 1 µl primer for both primers, 10 µl deion-
Finding
0-3
(n=20)
Homogenous vaginal 0 (0)
discharge
Vaginal pH>4.5 3 (15)
Amine positive 0 (0)
Nogent Score P value
4-6 7-10
(n=44) (n=38)
29 (65) 31 (81) 0/0009
29 (65) 32 (84) 0/03
24 (54) 38 (100) 0/0001
ized water, and 4µl of the template DNA.
The amplification protocol was as follow; Initial de-
naturation at 95˚C for 4 minutes followed by 25 cycles of
95˚C, 55˚C and 72˚C for 1 Minute each, with a final exten-
sion at 72˚C for 7 minutes. Following addition of DNA Safe
StainTM, the PCR product was electro phrased in 1% aga-
rose gel for 1 hour at 90 V and detected by UV trans illumi-
nation (wavelength 254 nm).
Statistical analysis
Data was statistically analyzed for association sig-
nificance of Atopobium vaginae with bacterial vaginosis and
Amsel criteria for diagnosing bacterial vaginosis using spss
program. Analysis was performed on MS Office Excel and P
value <0.05 was taken as significant.
RESULTS
A total of 102 vaginal swabs from women with symp-
toms of abnormal vaginal odor or discharge, and also 30
urine samples from women with urinary tract infection and
30 urine samples from healthy pregnant women were regis-
tered in this cross-sectional study. All subjects were within
18-35 years of age. In the 102 vaginal samples, 37% (38/102)
were found as indicative of BV based on the Amsel criteria
[14,15,16,17]. Forty three percent (44/102) were detected as
"intermediate vaginal flora". Twenty of the asymptomatic
patients had only one or two out of the four clinical crite-
ria and no Atopobium vaginae was found, neither by culture
or PCR method. Ten percent (4/44) of the Atopobium vagi-
nae were isolated from patients with "intermediate normal
flora". The PCR product was subsequently sequenced and
following analysis of the sequence by chromas Program, it
was confirmed to be related to Atopobium vaginae as shown
in Fig.1.
Clue cells 1 (5) 7 (15) 27 (71) 0/003
Prevalence of Atopo- 0(0) 4 (10) 25 (65) 0/0007
bium vaginae
Out of the 38 total BV cases, 66% (25/38) Atopobium va-
ginae were isolated. However, none was isolated from the as-
ymptomatic cases. From the 30 urine samples from women
with urinary tract infection, 10% (3/30) Atopobium vagi-
nae were isolated. None were isolated from the urine samples
of healthy pregnant women. The picture of electrophoresed
agarose gel showing the PCR protocol performed directly on
the vaginal discharges was shown in Figure 2.
Figure 2. Species-specific PCR assay for the detection of Atopobi-
um vaginae in vaginal and urine samples (agarose gel-electroforesis
of PCR products of the 16S rRNA gene). The product size is 155
bp. From right to left: 1kb Ladder, control positive, control negative
and positive samples.
45 N. Amirmozafari et al /JABS, 10 (3): 43-46, 2016
DISCUSSION
Bacterial vaginosis is a risk factor for obstetric infec-
tions and is a cause of mal odorous vaginal secretions. It is
linked to various adverse outcomes in women and causes
significant gynecological and obstetric morbidity such as
preterm birth, sexually transmitted disease, infections fol-
lowing gynecological surgery and pelvic inflammatory dis-
ease (PID) [1,3,4].Vaginal lactobacilli can prevent coloniza-
tion by and growth of other potentially pathogenic bacteria;
thereby, reducing the risk of developing BV.
Progression of a reasonable tool for identification of BV
is crucial. There is not any standard, credible and objec-
tive assay for rapid diagnosis of BV. The Amsel criteria and
the Nogent score are the two presently attainable diagnostic
procedures and are often used in most studies. Most studies
which were based on Nugent score, expressed some limita-
tions. It must be performed on a fresh swab, and any post-
ponement in transporting the swab makes the test difficult
to perform. It is rarely used by physicians due to the time
it takes to read the slides and the need for a skilled techni-
cian for interpretation. Additionally, pathogens associated
with BV are not identified by such a techniqueas is the case
for Atopobium vaginae [18]. Recent studies have shown that
molecular amplification techniques have both higher sensi-
tivity and specificity for the diagnosis of BV[18,19,20,21]
compared to the Nugent score.A few studies examined the
association between BV and vaginal bacterial species by
molecular methods[22,23,24,25]. The results of this investi-
gation are in agreement with previous molecular approaches
that reported a high load of Atopobium vaginae in most pa-
tients with BV.
It is estimated that 20-30 % of women in reproductive
age who refer to sexually transmitted disease (STD) clinics
suffer from BV; however, its prevalence can be as high as 50-
60 % in high-risk populations[4,9,10].Prevalence of bacte-
rial vaginosis in this study was around 37 %. The prevalence
rates of BV were reported to be 31% by Abbai et al in 2015
[11], 39% by Bradshaw et al in 2005 [12], 40% by Mitchellet
al in 2012 [13], and 38% by Vicky et al in 2014 [14]. Cross-
sectional studies have reported that Atopobium vaginae was
present in 0-20% of women with normal vaginal flora and in
50-78% of women with BV [15,16]. In this investigation, the
isolation rate of Atopobium vaginae in women with BV was
66% (25/38). Fredricks and Marrazo reported the sensitiv-
ity and specificity of Atopobium vaginae for BV to be 96%
and 80%, respectively [17]. This bacterium was detected by
PCR in 96% of patients with BV [26]. Our data also clearly
confirm the involvement of Atopobium vaginae in BV. This
bacterium is a Gram-positive anaerobic microbe which can-
not be easily isolated by classical microbiological methods.
It is hardly detected in healthy women vaginal fluid but is
commonly found in patients with BV. This bacterium was
isolated with different frequencies such as 50% according
to Burton et al[27], 70% according to Ferris et al [28], and
more than 95% according to Verhelst et al. and Verstraelen
et al [16].
It seems as though, measurement of Atopobium vaginae
bacterial loadis a good predictor for BV. However, Bradshaw
et al[12]claimed thatthe shear detection of Atopobium vagi-
nae can be more forecasting of BV than the bacterial load.
This difference between our study and that of Bradshaw et
al. may be linked to differences in epidemiological charac-
teristics and/or PCR assays. Geographical and/or ethnic ori-
gin, pregnancy status, risks of STDs, and prevalence of BV
in the population studied by Bradshaw et al. [12]were shown
to influence the rate of Atopobium vaginae in the vaginal
flora. Additionally, PCR technical parameters such as DNA
target, directly influence the sensitivity of PCR assays. The
16S rRNA target used in this study for Atopobium vaginae
(length, 155 base pairs) was shorter than that used by Brad-
shaw et al (length, 430 base pairs).It can be hypothesized
that our molecular tool is more sensitive.
In another study, Burton et al. [27] used a different set
of Atopobium vaginae specific PCR primers and detected it
in 50% of Canadian BV patients. Probably the most mean-
ingful observation in the present study and that of Burton et
al. [27]was that no Atopobium vaginae was detected in the
asymptomatic subjects. The prevalence of Atopobium vagi-
nae reported by various workers varies from 50-78%. The
difference may be due to the fact that different authors have
studied different types of population and have regarded dif-
ferent criteria for selecting the cases of BV [25].
This is the first report for detection of Atopobium vaginae
associated BV in Iranian women. Although PCR seems to
be the most sensitive method for detection of Atopobium-
vaginae. Gram staining can complement the PCR results.
The high prevalence of these bacteria reported in this study
in Iranian young women can be very concerning, since BV
raise women susceptibility to HPV, HIV and other important
sexually transmitted diseases. For this, BV has to be proper-
ly and urgently diagnosed in order to be sufficiently treated.
Acknowledgements
This investigation was financially supported by a gen-
erous grant (No. 25072-30-03-93) from Iran University of
Medical Sciences.
REFERENCES
[1] Abbai N, Reddy T, Ramjee G. 2015. Prevalent bac-
terial vaginosis infection-a risk factor for incident sexually
transmitted infections in women in durban, south africa. In-
ternational journal of STD & AIDS. 0956462415616038.
[2] Baruah F, Sharma A, Das C, Hazarika N, Hussain J.
2014. Role of gardnerella vaginalis as an etiological agent
of bacterial vaginosis. Iranian journal of microbiology. 6(6):
409.
[3] Bautista, C.T., E. Wurapa, W.B. Sateren, S. Morris,
B. Hollingsworth and J.L. Sanchez, 2016. Bacterial vagino-
sis: A synthesis of the literature on etiology, prevalence, risk
factors, and relationship with chlamydia and gonorrhea in-
fections. Military Medical Research. 3(1): 1.
[4] Bradshaw C, Tabrizi S, Fairley C, Morton A, Rud-
land E, Garland S. 2006. The association of Atopobium va-
ginae and Gardnerella vaginalis with bacterial vaginosis and
recurrence after oral metronidazole therapy. Journal of In-
fectious Diseases. 194(6): 828-836.
[5] Bretelle F, Fenollar F, Baumstarck K, Fortanier C,
Cocallemen J, Serazin V, Raoult D, Auquier P, Loubière S.
2015. Screen-and-treat program by point-of-care of Atopo-
bium vaginae and Gardnerella vaginalis in preventing pre-
term birth (autop trial): Study protocol for a randomized
controlled trial. Trials. 16(1): 1.
[6] Burton J, Devillad E, Cadieux P, Hammond J, Reid
G. 2004. Detection of Atopobium vaginae in postmenopausal
women by cultivation-independent methods warrants fur-
ther investigation. Journal of Clinical Microbiology. 42(4):
1829-1831.
[7] Burton J, Reid G. 2002. Evaluation of the bacterial
46
N. Amirmozafari et al /JABS, 10 (3): 43-46, 2016
vaginal flora of 20 postmenopausal women by direct (nu-
gent score) and molecular (polymerase chain reaction and
denaturing gradient gel electrophoresis) techniques. Journal of
Infectious Diseases. 186(12): 1770-1780.
[8] Cartwright C, Lembke B, Ramachandran K, Body
B, Nye M, Rivers C, Schwebke J. 2012. Development and
validation of a semiquantitative, multitarget pcr assay for di-
agnosis of bacterial vaginosis. Journal of Clinical Microbiol-
ogy. 50(7): 2321-2329.
[9] Cox C, Watt A, McKenna J, Coyle P. 2016. Myco-
plasma hominis and Gardnerella vaginalis display a signifi-
cant synergistic relationship in bacterial vaginosis. Euro-
pean journal of clinical microbiology & infectious diseases.
35(3): 481-487.
[10] Ferris M, Masztal A,Martin D. 2004. Use of spe-
cies-directed 16s rrna gene pcr primers for detection of atop-
obium vaginae in patients with bacterial vaginosis. Journal
of Clinical Microbiology. 42(12): 5892-5894.
[11] Fethers K, Twin J, Fairley C, Fowkes F, Garland
S, Fehler G, Morton A, Hocking J, Tabrizi S, Bradshaw C.
2012. Bacterial vaginosis (bv) candidate bacteria: Associa-
tions with bv and behavioural practices in sexually-experi-
enced and inexperienced women. PLoS One. 7(2): e30633.
[12] Fredricks D, Fiedler T Marrazzo J. 2005. Molecular
identification of bacteria associated with bacterial vaginosis.
New England Journal of Medicine, 353(18): 1899-1911.
[13] Fredricks D, Marrazzo J. 2005. Molecular meth-
odology in determining vaginal flora in health and disease:
Its time has come. Current infectious disease reports. 7(6):
463-470.
[14] Gergova R, Strateva T, Mitov I. 2013. Gardnerella
vaginalis-associated bacterial vaginosis in bulgarian women.
The Brazilian Journal of infectious diseases. 17(3): 313-318.
[15] Jespers V, Crucitti T, Menten J, Verhelst R, Mwaura
M, Mandaliya K, Ndayisaba G, Delany-Moretlwe S, Ver-
straelen H, Hardy L. 2014. Prevalence and correlates of
bacterial vaginosis in different sub-populations of women
in sub-saharan africa: A cross-sectional study. PLoS One.
9(10): e109670.
[16] Livengood C. 2009. Bacterial vaginosis: An over-
view for 2009. Rev Obstet Gynecol. 2(1): 28-37.
[17] Marrazzo J, Antonio M, Agnew K, Hillier S. 2009.
Distribution of genital lactobacillus strains shared by female
sex partners. Journal of Infectious Diseases. 199(5): 680-
683.
[18] Marrazzo J, Koutsky L, Eschenbach D, Agnew K,
Stine K, Hillier S. 2002. Characterization of vaginal flora
and bacterial vaginosis in women who have sex with wom-
en. Journal of Infectious Diseases. 185(9): 1307-1313.
[19] Marrazzo J, Thomas K, Fiedler T, Ringwood K,
Fredricks D. 2008. Relationship of specific vaginal bacte-
ria and bacterial vaginosis treatment failure in women who
have sex with women. Annals of internal medicine. 149(1):
20-28.
[20] Menard J, Fenollar F, Henry M, Bretelle F, Raoult
D. 2008. Molecular quantification of Gardnerella vaginalis
and Atopobium vaginae loads to predict bacterial vaginosis.
Clinical infectious diseases. 47(1): 33-43.
[21] Mitchell C, Manhart L, Thomas K, Fiedler T, Fred-
ricks D, Marrazzo J. 2012. Behavioral predictors of coloni-
zation with lactobacillus crispatus or lactobacillus jensenii
after treatment for bacterial vaginosis: A cohort study. In-
fectious diseases in obstetrics and gynecology.2012:706540.
[22] Onderdonk A, Delaney M, Fichorova R.2016. The
human microbiome during bacterial vaginosis. Clinical mi-
crobiology reviews. 29(2): 223-238.
[23] Schuyler J, Mordechai E, Adelson M, Gygax S, Hil-
bert D. 2015. Draft genome sequence of a metronidazole-
susceptible Atopobium vaginae isolate. Genome announce-
ments. 3(5): e00991-00915.
[24] Shipitsyna E, Roos A, Datcu R, Hallén A, Fredlund
H, Jensen J, Engstrand L, Unemo M. 2013. Composition of
the vaginal microbiota in women of reproductive age-sensi-
tive and specific molecular diagnosis of bacterial vaginosis is
possible? PLoS One. 8(4): e60670.
[25] Tabrizi S, Fairley C, Bradshaw C, Garland S. 2006.
Prevalence of Gardnerella vaginalis and Atopobium vaginae
in virginal women. Sexually transmitted diseases. 33(11):
663-665.
[26] Verstraele H, Verhelst R, Claeys G, Temmerman
M, Vaneechoutte M, 2004. Culture-independent analysis of
vaginal microflora: The unrecognized association of Atopo-
bium vaginae with bacterial vaginosis. American journal of
obstetrics and gynecology. 191(4): 1130-1132.
[27] Vieira-Baptista P, Lima-Silva J, Pinto C, Saldanha
C, Beires J, Martinez-de-Oliveira J, Donders G. 2016. Bac-
terial vaginosis, aerobic vaginitis, vaginal inflammation and
major pap smear abnormalities. European journal of clinical
microbiology & infectious diseases. 35(4): 657-664.